PLEK2 functions as a co-factor of YTHDF2 to enhance TYMS mRNA stability in colorectal cancer

High expression of nucleotide synthetic enzyme thymidylate synthase (TYMS) is responsible for the resistance to fluorouracil (FU) treatment and worse survival in colorectal cancer. Herein, we revealed that Pleckstrin-2 (PLEK2), highly expressed in CRC, enhanced TYMS mRNA stability via its interaction with YTHDF2 in a m6A-dependent manner. Silencing of PLEK2 led to the downregulation of TYMS, and consequent inhibition of CRC cell proliferation via the cellular senescence. Additionally, PLEK2 is also required for CRC cell migration, invasion and stemness-like properties. PLEK2 inhibition is sufficient to ameliorate the progression of CRC. Moreover, loss of PLEK2 inhibited AOM/DSS-induced colonic tumorigenesis in vivo. Together, our study identified PLEK2 as a key regulator for the progress of CRC via the regulation of TYMS expression, and demonstrated that PLEK2 is a novel therapeutic target for CRC. Overall design: 1X107 HCT116 cells transduced with retroviruses encoding HA tagged PLEK2 and empty vector were collected and lysed according to the instructions of RIP Immunoprecipitation Kit (Geneseed, P0101). Briefly, Agarose beads coated with 2µg of HA antibodies against human PLEK2 were incubated with prepared cell lysates overnight at 4°C. RNA was eluted by 15 µL of RNase-free water in the following steps, then ribominus rRNA were depleted by the rRNA depletion kits (Thermo, K155001), for high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and applied to Illumina NovaSeq 6000 system for 150 nt paired-end sequencing (Novogene).

核苷酸合成酶胸苷酸合酶（thymidylate synthase, TYMS）高表达是结直肠癌患者对氟尿嘧啶（fluorouracil, FU）治疗产生耐药性及预后不良的关键原因。本研究证实，在结直肠癌（colorectal cancer, CRC）中高表达的pleckstrin-2（PLEK2）可通过与YTHDF2相互作用，以m6A依赖的方式增强TYMS mRNA的稳定性。敲低PLEK2可下调TYMS的表达，并通过诱导细胞衰老进而抑制结直肠癌细胞的增殖。此外，PLEK2同样是结直肠癌细胞迁移、侵袭及干细胞样特性维持所必需的。抑制PLEK2即可有效阻滞结直肠癌的进展。进一步体内实验表明，敲除PLEK2可抑制AOM/DSS诱导的小鼠结肠肿瘤发生。综上，本研究鉴定PLEK2为通过调控TYMS表达参与结直肠癌进展的关键调控因子，并证实PLEK2是结直肠癌的新型治疗靶点。

整体实验设计：收集1×10^7个转染了携带HA标签PLEK2的逆转录病毒及空载体的HCT116细胞，按照RIP免疫沉淀试剂盒（Geneseed, P0101）的说明书进行细胞裂解与后续操作。具体步骤为：将包被有2μg抗人PLEK2的HA抗体的琼脂糖微球与制备好的细胞裂解液在4℃下孵育过夜。后续使用15μL无RNase水洗脱RNA，再通过rRNA去除试剂盒（Thermo, K155001）去除核糖体RNA。为开展高通量测序（high-throughput sequencing），按照试剂盒说明书构建测序文库，并将文库置于Illumina NovaSeq 6000平台进行150nt双端测序（诺禾致源, Novogene）。